Outbreak of avian mycobacteriosis in flocks of domestic pigeons: An epidemiological approach.

Background and Objectives Pigeons are extensively kept for homing and racing purposes in Iran. The main objective of this study was to investigate dissemination of M. avium subsp. avium (MAA) in pigeon aviaries in Tabriz, North-western Iran. Materials and Methods Postmortem pathologic specimens from thirty-nine out of 140 birds collected from private flocks (n=3), were subjected to bacterial culture out of which 3-4 mycobacterial isolates were recovered. Results Applying a five-PCR diagnostic algorithm targeting short but definitive stretches of 16S rRNA and RV0577 genes, IS6110, IS901 and IS1245 genomic loci, proved all the isolates were MAA. They were either IS901+/IS1245+(n=22) or IS901+/IS1245- (n=12). When four healthy cattle sensitized against Mycobacterium bovis AN5 and Mycobacterium avium D4 were tuberculinated, the results confirmed the observed skin reactions against bovine tuberculin in animals sensitized with M. avium were large enough to complicate test interpretation. Conclusion We believe the extent of such epidemiological impact deserves further investigation if progress in control of bovine tuberculosis is intended.

With characterization of species-specific genomic markers and introduction of PCR and RFLP, it is now possible to differentiate MAC bacteria as IS900 in MAP (8,9), IS901/902 in MAA as well as MAS (10, 4), IS1245 in MAH and some strains of MAA (11,12), IS1642 in MAA (13) and finally FR300 in some MAA strains (10,14) have been shown to be speciesspecific.
Avian tuberculosis has been reported in numerous species of pet, zoo, wild-life and avicultural birds (15, 7) including ring-neck doves (Streptopelia risoria)

MATERIALS AND METHODS
Following complaints from three pigeon fanciers that many of their birds showed poor physical condition and unusual behavior for sometime e.g. cachexia, depression, no interest to fly, poor appetite and weight loss, these colonies were visited by the local veterinary officer and birds in their nests were closely examined. In consequence, 39 pigeons were collectively selected for postmortem examination on the grounds of their poor health condition. Birds were transferred to the diagnostic laboratory where, they were euthanized and necropsied. Microscopic slides were subsequently prepared from tissue impressions of the pathological lesions stained with Ziehl-Nielson technique in search for acid-fast bacilli (AFB). Using sterile material and equipments, all the associated specimens for each bird were pooled and grinded in a pestle and mortar containing sand. These included lesions found in liver, spleen, heart, gut, musculoskeletal system as well as gonads. The homogenized mixture was exposed to a cocktail of 5 ml N-acetyl-L-cysteine (5 g/l), 5 ml of sodium hydroxide (2 g/l) and 0.01 ml of sodium citrate solution for 15 min (20) in order to remove bacterial contaminators. About 5 ml of the supernatant was added to equal amount of phosphate buffer and centrifuged (3, 500 g/15 m). Again, 0.5 ml phosphate buffer was added to the deposit and the mixture was stirred to make the inoculation suspension. The inoculums were cultured on 4 culture slopes including glycerinated Lowenstein-Jensen (LJG) medium, pyruvate-enriched Lowenstein-Jensen medium (LJP), mycobactin J-supplemented Herrold-egg yolk medium and plain Herrold-egg yolk medium. The inoculated slopes were incubated at 37ºC for 8 weeks.

Molecular identification.
Each isolate was sub-cultured onto two fresh LJG slants in order to achieve bacterial growth enough for extraction of  (Table 1). PCRs were conducted as described elsewhere with incorporation of positive (Mycobacterium bovis AN5 and Mycobacterium avium subsp. avium D4 strains) and negative (double-distilled water) controls (10, 11, 23). Analysis of PCR amplicons was conducted on ethidium bromide-stained 2% agarose gels in a submerged electrophoresis system.

Sensitization/skin test experiments.
In the next phase, 4 healthy Holstein-Friesian cows in two two-animal groups were deep-intramuscularly administrated 0.5 ml fine-powdered heat-inactivated bacterial mass of Mycobacterium bovis AN5 and Mycobacterium avium subsp avium D4 strains respectively suspended in paraffin in order to immunologically sensitize them. Twelve weeks after initial injections, bovids were tuberculinated intradermally with 10,000 and 2,500 IU of bovine and avian PPD tuberculins respectively administrated in their left neck as instructed by the Iranian Veterinary Organization (IVO) in the national test-and-slaughter scheme. The experiment was further repeated for three times on three-cattle groups each included one bovid sensitized to M. bovis, one animal sensitized to M. avium and the last one non-sensitized as control.

RESULTS
In necropsy, characteristic AT granulomas were observed in internal organs, e.g. liver, spleen, heart, gut, kidney, ovaries, testes, eyes and musculoskeletal system specifically in legs, sternum and pectoral muscle of all the birds. Lesions in liver were particularly observable as 18 birds carried such lesions. In bacteriology, out of the 39 necropsied pigeons submitted for the test, 35 isolates were recovered all of which showed characteristic morphology of the MAC in bacterial culture and microscopy. The genomic material, however, was available from 34 isolates for molecular identification experiments. In 16S rRNA test, all the isolates produced a 543bp PCR fragment, an indication that they belonged to Mycobacterium genus ( Table 1). None of the isolates in the study setting produced the 870bp amplicon in RV0577 assay confirming they were not member of M. tuberculosis complex (Table 1). In IS901-PCR experiment all the isolates produced an amplicon as large as 1,084 bp (Fig.  1B). This observation confirmed they carried the insertion sequence and therefore were MAA isolates. In IS1245-PCR experiment, 9 isolates produced the characteristic 427bp length amplicon (Fig. 1A).
In sensitization/skin test experiments, with no exception, all the sensitized cattle showed skin reactions towards bovine tuberculin although in those cattle sensitized to MAA D4 such reactions were overshadowed by the size of skin reactions against avian tuberculin. Nevertheless, they also produced a considerably large reaction to the bovine tuberculin.

DISCUSSION
The present study explains naturally occurring mycobacteriosis of pigeons in Iran. There are a    Such isolates that belong to serotypes 1, 2 and 3 of MAA are considered as the most pathogenic strains of MAA in birds as well as humans (4, 28). Our observation that numerous gross tuberculous lesions were detected in internal organs of birds resembled the same postmortem scenario reported by other authors (29) but seems to conflict with findings of others that believe that columbiforms tubercles do not normally develop (30). This controversy might be explained by factors linked to the pigeon breed and also the strain pathogenicity.
Our sensitization test in cattle showed MAA had the potential to induce hypersensitivity reactions in the intradermal tuberculin test skin and affect the results interpretation. In 2007, some 50,000 bovids were tuberculinated in East Azerbaijan province where the present study conducted. This resulted in removing over 200 animals out of which 71 produced non-definitive test result. Mycobacterium bovis was apparently the main infecting agent of all these animals but no information is available on frequency of MAA infection in these bovids. Cattle are not normally sensitive to MAA but infection of digestive system in these animals via consumption of feedstuff has been previously reported (26). In an earlier comprehensive study by the Iranian Veterinary Organization (IVO), MAA isolates were isolated from lymph nodes of cattle with a positive test result. Pigeons are frequent visitors of cattle farms around their colonies where they search for food and water and also release their droppings thus infecting the environment and exposing cattle to pathogenic MAA.
In conclusion, we suggest further molecularepidemiology studies in order to detect pathogenic strain of MAA in cattle and tracing the source(s) of MAA in the environment.